Abstract

The p21-activated kinase (PAK) family members are key effectors of Rho family GTPases. These small G protein act as regulatory switches that control such cellular processes as motility, proliferation, and cell survival. Some members of this family (such as Cdc42) have been shown to be required for Ras driven tumorigenesis. PAK4 is a key effector for Cdc42 and mediates downstream signals that control cell motility, proliferation and cell survival. To date, PAK4 is the only PAK family member that has been shown to be oncogenic and able to drive anchorage independent growth when activated. PAK4 expression and activity is broadly up-regulated in solid tumors of epithelial origin and the PAK4 gene maps to an amplicon associated with ovarian and pancreatic cancers. The PAK family consists of two sub groups; group I that consists of PAK1,2,3 and group II that consists of PAK4,5,6. Both PAK4 and PAK1 have been shown to be required for Ras driven transformation. PF-3758309 is an ATP-competitive inhibitor of PAK4 kinase domain (Kd = 4.5 nM). In engineered cell assays, PF-3758309 inhibited PAK4 dependent phosphorylation of its substrate GEF-H1 (IC50 = 1 nM). Anchorage-independent growth inhibition is a critical phenotype that has been rigorously associated with PAK4 inhibition. PF-3758309 potently inhibits the anchorage independent growth of HCT116 cells (IC50 = 0.24 nM). PF-3758309 exhibited broad anti-proliferative activity across a panel of 67 cell lines (CRC/pancreatic/NSCLC): 66% IC50 < 100 nM and 36% IC50 < 10 nM. Global cellular biological analysis has been used to show that PF-3758309 potently modulates signaling nodes expected to be dependent of PAK4: PAK4/cdc42, PAK4/cofilin, LIMK/cofilin, and ARF/\#947;COP. In human a xenograft tumor model, PF-3758309 demonstrated a dose-dependent inhibition of both GEF-H1 and PAK4 phosphorylation. At efficacious exposures of PF-3758309, significant inhibition of 3\#8217;-deoxy-3\#8217;-fluorothymidine (FLT) uptake was observed in tumors but not in the control tissue (liver). Tumor proteomic studies have revealed that PF-3758309 modulates pathways, as expected for an inhibitor of PAK, in a dose-dependent manner. PF-3758309 is highly efficacious in human xenograft tumor models. Five of seven models tested showed robust tumor growth inhibition (>70%TGI at 15-20 mg/kg PO) by PF-3758309: HCT116, A549, MDAMB231, M24met, and Colo205. PK/PD modeling of tumor growth inhibition in the HCT116 model yielded an EC50 = 400 pM (unbound). PF-3758309 is a kinase inhibitor that has been widely profiled biochemically: (146 kinase assays, 74 CEREP assays), cellularly (171 cellular assays), and in vivo (tumor proteomics). By biochemical analysis, PF-3758309 is an inhibitor of PAK1, 4, 5, 6. Broad kinase screening has demonstrated that this is a selective inhibitor with potential additional activities (Src, Fyn, AMPK, CHK). The pharmacodynamic and antitumor effects of PF-3758309 support its evaluation as an anticancer agent.